In vitro cultures of animal cells are hosts for an increasing assortment of recombinant protein products. This is a relatively new phenomenon, beginning in the mid 1980's. In this short period of time, these proteins have made a substantial contribution to the U.S. economic market. In the pharmaceutical industry alone, biotherapies, which are primarily recombinant proteins, generated $1.2 billion in U.S. sales in 1991 and may grow to nearly $8.0 billion in 2001 as more proteins become commercially available [Thayer, A. M., C & EN, Feb. 25, 27 (1991)]. The market size and its capacity for growth demonstrate the importance of recombinant protein production to the fields of biotechnology and bioengineering.
Recombinant proteins derived from animal cells are employed in a range of disciplines from agriculture to medicine to basic research. During the past decade in medicine, progress has been made in genetic engineering and animal-cell cultivation to produce an increasing assortment of recombinant proteins in sufficient quantities for the development of new drug therapies. The proteins themselves can serve as therapies, or they can be used to identify and test chemotherapies that regulate protein function in vivo. Recombinant tissue plasminogen activator is an example of a protein therapy. It is given to patients with evolving myocardial infarction to dissolve coronary thrombus and restore blood flow to the ischemic area [Longridge, D. J., Foilenfant, M. J., Maxwell, M. P., Ford, A. J., and Hughes B., Cardiovasc. Res., 24, 697 (1990)]. In clinical trials, another protein therapy, erythropoietin, has been used to treat anemia in patients with progressive renal failure [Eschbach, J. W., Kelly, M. R., Haley, N. R., Abels, R. I., and Adamson, J. W., N. Engl. J. Med., 321, 158 (1989)] and sickle cell disease [Goldberg, M. A., Brugnara, C., Dover, G. J., Schapira, L., Charache, S., and Bunn, H. F., N. Engl. J. Med., 323, 366 (1990)]. Examples of protein-regulating chemotherapies include enalapril, an inhibitor of angiotensin-converting enzyme, for the treatment of sickle cell disease. [Falk, R. J., Scheinman, J., Phillips, G., Orringer, E., Johnson, A., and Jennette, J. C., N. Engi. J. Med., 326, 910-915 (1992)]. A chemotherapy derived from azole regulated cytochrome P-450 dependent testosterone biosynthesis [Bossche, H. V., Lauwers W., Willemsens, G., Cools, W., Prog. Clin. Bioi. Res., 185A, 187 (1985)].
At present, both procaryotes and eucaryotes are utilized as hosts for commercial production of recombinant proteins. The choice of one over the other is based on the structural complexity of the protein being produced and the desired yield. If a protein can be produced in a biologically active form from either host, procaryotes are preferred: they grow faster and express more protein than animal cells [Marino, M. H., BioPharm, July/August, 18 (1989); Bebbington, C. and Hentschel, C., Trends Biotechnol., 3, 314 (1985)]. Doubling times are in hours rather than days. Similarly, yields are in grams of protein per liter media rather than milligrams per liter. Eucaryotes are chosen as hosts when procaryotes are unable to produce functional protein [Marino, M. H., BioPharm, July/August, 18 (1989); Bebbington, C. and Hentschel, C., Trends Biotechnol., 3, 314 (1985)]. This typically occurs when the protein requires post-translational modification (e.g., glycosylation, phosphorylation or macromolecular assembly) to be functional. Bacteria cannot perform post-translational modifications at all; simple eucaryotes such as yeast do so to a limited extent; but complex eucaryotes such as animal cells, with few exceptions, perform the entire complement of post-translational modifications.
Commercial production of recombinant proteins from animal cells requires that the production process be reliable, yielding consistent amounts of product with reproducible biological activity. Such stringency has been achieved in vitro from animal cells cultured in a bioreactor which provides a controlled environment for cell growth.
Several bioreactor designs have been employed in the past for the cultivation of animal cells [Merten, 0. W., Trends Biotechnoi., 5, 230 (1987); Nelson, K. L., BioPharm., February, 42 (1988); Tramper, J., Smit, D., Straatman, J., and Vlak, J. M., Bioprocess Eng., 3, 37 (1988)]. These include a stirred-tank reactor, a hollow-fiber reactor containing porous fiber bundles in which cells grow, and an airlift reactor in which gas bubbles rise through a draft tube lifting the culture fluid to the top of the reactor where it returns to the bottom through the annular space between the draft tube and outer shell of the reactor. [See Inlow et al., U.S. Pat. No. 5,024,947, entitled "SerumFree Media For the Growth on Insect Cells and Expression of Products Thereby", issued Jun. 18, 1991]. Bioreactors up to 10,000 liters in size are used in industry for animal cells [Rhodes, M., Genetic Engineering News, 10, March, 7 (1990)]. Choosing the size and type of bioreactor for a particular process depends on a number of factors such as market demand, cell properties and yield. Hollow-fiber reactors are currently limited to volumes on the order of one liter [Cracauer et al., U.S. Pat. No., 4,804,628, entitled "Hollow Fiber Cell Culture Device and Method of Operation" issued Feb. 14, 1989; Nelson, K. L., BioPharm., March, 34 (1988)]. Airlift reactors can not readily keep microcarrier cultures (attachment-dependent cells growing bound to beads that are typically 100 .mu.m in diameter) well mixed [Merten, O. W., Trends Biotechnol., 5,230 (1987)]. The stirred-tank reactor is the system of choice for many companies because of its flexibility: it can support the growth of both suspension and anchorage-dependent cells, can be operated in different feed modes, and can be scaled up to very large volumes (10,000 liters) [Nelson, K. L., BioPharm., March, 34 (1988); Nelson, K. L., BioPharm., February, 42 (1988)].
In bioreactors, mixing is essential for cell proliferation: it supplies cells with nutrients and oxygen, maintains a homogenous environment throughout the reactor, and prevents cells from settling. But in conventional bioreactors, mixing can also cause cell damage from sufficiently large hydrodynamic forces. In a stirred tank, for example, cell damage has been attributed to two mixing phenomena: bulk-fluid turbulence and gas/liquid interfaces [O'Connor, K. C. and Papoutsakis, E. T., Biotechnol. Tech., 6, 323 (1992); Kunas, K. T. and Papoutsakis, E. T., Biotechnol. Bioeng., 36, 476-483 (1990)]. Typically, these interfaces arise during cultivation as a result of sparging, vortex formation, turbulent eddies, fluid-wall shear gradients and surface oxygenation. Cell damage from bulk-liquid turbulence has been correlated to the ratio of the Kolmogorov-scale eddy size to bead diameter for microcarrier cultures [Cherry, R. S. and Papoutsakis, E. T., Bioprocess Eng., 1, 29-41 (1986); Croughan, M. S., Hamel, J. F., and Wang, D. I. C., Biotechnol. Bioeng., 29, 130-141 (1987)] and cell diameter for suspension cultures [Kunas, K. T. and Papoutsakis, E. T., Biotechnol. Bioeng., 36, 476-483 (1990); McQueen, A., Meilhoc, E., and Bailey, J. E., Biotechnol. Letters, 9, 831 (1987)]. Damage initiates as the ratio approaches unity and intensifies at lower values. It has been proposed that eddies of the same size or smaller than cell particles (microcarrier beads or individual cells) cause high shear stresses on the cell surface, interparticle collisions and reactor-particle collisions [Cherry, R. S. and Papoutsakis, E. T., Bioprocess Eng., 1, 29-41 (1986)]. For larger eddies, shearing and collisions are minimized as cell particles move in eddy streamlines.
Because animal cells are not enclosed in a cell wall like bacteria, they are susceptible to hydrodynamic forces within a bioreactor. Agitation, shear and other hydrodynamic phenomena have a profound effect on cell morphology and physiology which can result in cell damage and death. From a morphological perspective, hydrodynamic forces alter cell shape, adhesion, membrane integrity and spreading [O'Rear, E. A., Udden, M. M., McIntire, L. V., and Lynch E. C., Biochim. Biophys. Acta, 691, 274 (1982); Lawrence, M. B., McIntire, L. V., and Eskin, S. G., Blood, 70, 1284 (1987); Ives, C. L., Eskin, S. G., and McIntire, L. V., In Vitro Cell. Dev. Biol. 22, 500 (1986)]. These morphological changes are accompanied by and related to physiological changes in DNA synthesis, mRNA synthesis and cytoskeletal rearrangement to name a few [O'Connor, K. C. and Papoutsakis, E. T., Biotechnoi. Tech., 6, 323 (1992); Diamond, S. L., Sharefkin, J. B., Dieffenbach, C., Frasier-Scott, D., McIntire, L. V., and Eskin, S. G., J. Cell. Physiol, 143, 364-371 (1990); Sato, M., Levesque, M. J., and Nerem, R. M., Arteriosclerosis, 7, 276 (1987)]. There are several mechanisms by which these hydrodynamic effects can cause cell death, including lysis from loss of membrane integrity, detachment of anchorage-dependent cells from surfaces, and reduced metabolic activity [O'Connor, K. C. and Papoutsakis, E. T., Biotechnol. Tech., 6, 323 (1992); Croughan, M. S., Hamel, J. F., and Wang, D. I. C., Biotechnol. Bioeng., 33, 731-744 (1989); Petersen, J. F., McIntire, L. V., and Papoutsakis, E. T., J. Biotechnol., 7, 229 (1988)].
Insect cells are distinctly different from animal cells. Very little is known about insect cell metabolism. There is a relatively small body of literature on the subject. Insects and vertebrate animals are classified in totally different phylogenetic systems. Consequently, insect cells have undergone a metabolic evolution that was completely independent compared to animal cell metabolic evolution. Insects have a unique life cycle and, as such, have distinct cellular properties. One of these is the lack of intracellular plasminogen activators in insect cells which are present in vertebrate cells. These differences were highlighted when investigators began to express recombinant intact human plasminogen in Sf9 cells after unsuccessful attempts in vertebrate cells. [Whitefleet-Smith, J., Rosen, E., McLinden, J., Ploplis, V. A., Fraser, M. J., Tomlinson, J. E., McLean, J. W., and Castellino, F. J., Arch. Biochem. Biophys., 271, 390 (1989)]. In the latter, plasminogen is rapidly converted by the activators to plasmin. Other differences include high expression levels of protein products ranging from 1 to greater than 500 mg/liter [Marino, M. H., BioPharm, July/August, 18 (1989)] and ease at which DNA can be cloned into the cells [Fraser, M. J., In Vitro Cell. Dev. Biol., 25, 225 (1989); Summers, M.D. and Smith, G. E., A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experiment Station Bulletin No. 1555 (Texas A & M University, College Station, Tex., 1988)], both of which make insect cells exceptional hosts for protein production.
Cultivation of insect cells is difficult particularly on a large scale or at a high cell density since they are more sensitive to hydrodynamic forces in a bioreactor [Wu, J., King, G., Daugulis, A. J., Faulkner, P., Bone, D. H., and Goosen, M. F. A., Appl. Microbiol. Biotechnol., 32, 249 (1989); Tramper, J., Williams, J. B., and Joustra, D., Enzyme Microb. Techno., 8, 33 (1986)] and have a greater oxygen uptake rate than most vertebrate cells [Maiorella, B., Inlow, D., Shauger, A., and Harano, D., Bio/Technology, 6, 1406 (1988)]. The oxygen uptake rate has been calculated to be 4.times.10.sup.-10 mole cell.sup.-1 hr.sup.-1 for Trichoplusia ni cells [Maiorella, B., Inlow, D., Shauger, A., and Harano, D., Bio/Technology, 6, 1406 (1988)] as compared to 0.1 to 2.times.10.sup.-10 mole cell.sup.-1 hr for typical vertebrate cells [Nelson, K. L., BioPharm., March, 34 (1988)]. Upon infection with baculovirus, the oxygen uptake rate for insect cells further increases by upwards of 30% [Kamen, A. A., Tom, R. L., Caron, A. W., Chavarie, C., Massie, B., and Archambault, J., Biotechnol. Bioeng., 38, 619 (1991)]. The sensitivity to hydrodynamic forces is so severe that growth of a Lepidopteran cell line, the fall armyworm ovary Spodoptera frugiperda (Sf9), is completely inhibited in bench-scale sparged stirred tanks or airlift bioreactors unless surfactant is added to protect the cell membrane [Murhammer, D. W. and Goochee, C. F., Biotechnol. Prog., 6, 391 (1990)].
The combination of enhanced respiration and hydrodynamic sensitivity places severe constraints on cell oxygenation: sufficient oxygen must be transported to the insect cells for respiration without inducing hydrodynamic damage to the cells. When the oxygen requirements of insect cells are not met, the culture is adversely affected. Under these conditions, the culture is hypoxic or anoxic. We have conducted extensive investigations of anoxic cultures of Sf9 cells [Hugler, W., "Protein Expression of Sf9 Cells Under Stress and Simulated Microgravity," Thesis, Tulane University (1994)]. Within the first three hours of anoxia, cell growth is arrested, total protein synthesis is reducedby 20%, and stress proteins are induced. If anoxia is continued for longer periods of time, cell death is evident. Others have observed that hypoxia reduces the yield of recombinant protein derived from insect cells by a factor of 5 [Scott, R.I., Blanchard, J. H., and Ferguson, C. H. R., Enzyme Microb. Technol., 14, 798 (1992)].
Several different types of bioreactors have been used for insect-cell cultivation. Most of them adequately address cell oxygenation but at the expense of the cell's hydrodynamic sensitivity. Stirred tanks typically oxygenate cells by sparging and impeller stirring of growth medium, generating hydrodynamic forces from both the mixing and bursting air bubbles at gas/liquid interfaces. Spin-filter adapters for stirred tanks provide bubble-free oxygenation, although substantial impeller stirring is still required [Avegerinos, G. C., Drapeau, D., Socolow, J. S., Mao, J.-I., Hsiao, K., and Broeze, R. J., Biotechnology, 8, 54 (1990)]. Airlift bioreactors are designed to eliminate hydrodynamic damage to the cells from impellers as mixing is achieved by rising gas bubbles [Inlow et al., U.S. Pat. No. 5,024,947, entitled "Serum Free Media For the Growth on Insect Cells and Expression of Products Thereby", issued June 18, 1991]. This design is flawed, however, in that by eliminating one source of hydrodynamic damage it increases another--air/liquid interfaces. The detrimental effect of impeller mixing and air/liquid interfaces on insect-cell culture has been documented [Murhammer, D. W. and Goochee, C. F., Biotechnol. Prog., 6, 391 (1990); Chalmers, J. J. and Bavarian, F. Biotechnol. Prog., 7, 151 (1991)].
Two reactor designs that consider the hydrodynamic sensitivity of insect cells are roller bottles and microencapsulation. In roller bottles, cells are mixed by the end-over-end rotation of the reactor instead of byan impeller [Inlow et al., U.S. Pat. No. 5,024,947, entitled "Serum Free Media For the Growth on Insect Cells and Expression of Products Thereby", issued Jun. 18, 1991]. In this environment, cells grow attached to the reactor wall rather than in suspension. As such, cell densities that are achieved in roller bottles are far less than in suspension cultures because growth is limited by the surface area of the reactor wall. In addition, roller bottles are not conducive to scale up to the large volumes required for commercial cultivation. Another approach to insect-cell cultivation is to encapsulate the cells within porous microbeads [King, G. A., Daugulis, A. J., Faulkner, P., Bayly, D., and Goosen, M. F. A., Biotechnol. Lett., 10, 683 (1988)]. This shields the cells from the hydrodynamic forces on the outside of the beads. The difficulty with this design is the possibility of cell aggregation and accumulation of waste products within the beads at high cell density. The wastes (e.g. ammonia and lactate) are byproducts of cell metabolism and adversely affect cell health at high concentrations [Petersen, J. F., McIntire, L. V., and Papoutsakis, E. T., Biotechnol., 7, 229 (1988)]. Cell aggregation is an important issue for both mammalian and insect cells where hydrodynamic forces are minimized. These cell types will frequently adhere to surfaces through proteins on the cell surface [Ben-Ze'ev, A., Farmer, S. R., and Penman, S. Cell., 21, 365 (1980)]. In slow turning vessels, cells can aggregate into structures several millimeters in diameter [Becker, J. L., Prewett, T. L., Spaulding, G. F., and Goodwin, T. J., J. Cell Biochem., 51, 382 (1993)]. The cores of such aggregates often become hypoxic or anoxic [Coleman, C. N. Semin. Oncol., 16, 169 (1989)]. For insect cells, oxygen deprivation of this type would lower recombinant proteins yields as described above.
Developing a bioreactor environment which achieves favorable conditions for insect-cell growth through a reduction in hydrodynamic forces is a topic of much interest today and is the focal point of this invention. The invention described here is a specific non-animal cell culture process that incorporates both reduced hydrodynamic forces and enhanced cell oxygenation. The culture is maintained as a single-cell suspension, instead of in an aggregated state. Waste byproducts are kept at lower concentrations than in conventional bioreactors. Combined these features create an environment that improves cultivation and fundamentally changes cell metabolism.
Much of the cultivation and production research on insect cells has been performed with Sf9 cells [Wu, J., King, G., Daugulis, A. J., Faulkner, P., Bone, D. H., and Goosen, M. F. A., Appl. Microbiol. Biotechnol., 32, 249 (1989); Maiorella, B., Inlow, D., Shauger, A., and Harano, D., Bio/Technology, 6, 1406 (1988); Godwin, G., Belisle, B., De Giovanni, A., Kohler, J., Gong, T., and Wojchowski, D., In Vitro, 25, 17a (1989)]. This cell line is frequently chosen because it grows more robustly than other insect cells, is an immortal cell line, can be adapted from attachment-dependent to attachment-independent growth, is an exceptional host for recombinant protein production as described in the following paragraph, can grow in serum-free media and is commercially available [Fraser, M. J., In Vitro Cell. Dev. Biol., 25, 225 (1989); Wu, J., King, G., Daugulis, A. J., Faulkner, P., Bone, D. H., and Goosen, M. F. A., Appl. Microbiol. Biotechnol., 32, 249 (1989); Godwin, G., Belisle, B., De Giovanni, A., Kohler, J., Gong, T., and Wojchowski, D., In Vitro, 25, 17a (1989)]. Individual Sf9 cells have diameters from 10 to 20 .mu.m and can be maintained by following standard published protocols known to those skilled in the art of animal cell culture with the following exceptions that are distinctly different from typical animal culture protocols: they grow optimally at 27 rather than 37.degree. C., external CO.sub.2 is not required for growth, and the optimal pH for growth media is 6.2 instead of 7.4 [Fraser, M. J., In Vitro Cell. Dev. Biol., 25, 225 (1989); Godwin, G., Belisle, B., De Giovanni, A., Kohler, J., Gong, T., and Wojchowski, D., In Vitro, 25, 17a (1989)].
Sf9 shows great promise as an animal-cell host for the production of recombinant proteins. One of the reasons is the ease at which proteins can be cloned, expressed and purified relative to vertebrate animal cells. Sf9 more readily accepts foreign genes coding for recombinant proteins than many vertebrate animal cells because it is very receptive to viral infection and replication [Bishop, D. H. L. and Possee, R. D., Adv. Gene Technol., 1, 55, (1990)]. Expression levels of recombinant proteins are extremely high in Sf9 and can approach 500 mg/liter [Webb, N. R. and Summers, M. D., Technique, 2, 173 (1990)]. The cell line performs a number of key post-translational modifications; however, they are not identical to those in vertebrates and, therefore, may alter protein function [Fraser, M. J., In Vitro Cell. Dev. Biol., 25, 225 (1989)]. Despite this, the majority of recombinant proteins that undergo post-translational modification in insect cells are immunologically and functionally similar to their native counterparts [Fraser, M. J., In Vitro Cell. Dev. Biol., 25, 225 (1989)]. In contrast to animal cell culture, Sf9 facilitates protein purification by expressing relatively low levels of proteases and having a high ratio of recombinant to native protein expression [Goswami, B. B. and Glazer, R. O. BioTechniques, 10, 626 (1991)].
There has been an explosive growth in the number of proteins that have been expressed in Sf9 with less than 10 by 1985 to over 100 by 1990. These include .beta.-interferon [Smith, G. E., Summers, M. D., and Fraser, M. J., Mol. Cell. Bioi., 3, 2156 (1983)], interleukin-2 [Smith, G. E., Ju, G., Ericson, B. L., Moschera, J., Lahm, H., Chizzonite, R., and Summers, M. D., Proc. Nat. Acad. Sci. USA, 82, 8404 (1985)], chimeric plasminogen activators [Devlin, J. J., Devlin, P. E., Clark, R., O'Rourke, E. C., Levenson, C., and Mark, D. F., Bio/Technology, 7, 286 (1989)]and macrophage colony stimulating factor [Chiou, C. J., and Wu, M. C., FEB, 259, 249 (1990)]to name a few.
Baculoviruses serve as expression systems for the production of recombinant proteins in insect cells. These viruses are pathogenic towards specific species of insects, causing cell lysis [Webb, N. R. and Summers, M. D., Technique, 2, 173 (1990)]. They are, and have been, a natural part of the ecosystem where they control the population size of their hosts [Miltenburger, H. G. and Kreig, A., Advances in Biotechnological Processes, 3, 291 (1984)]. Some 300 species of baculovirus have been isolated. They are nonhazardous to humans, other vertebrates and indeed most invertebrates. After acute exposure of baculovirus from Autographa californica, Mamestra brassicae and Cydia pomonella, NMRI mice and Chinese hamsters had no chromosomal aberrations or health disturbances [Miltenburger, H. G. and Kreig, A., Advances in Biotechnological Processes, 3, 291 (1984)]. Similarly, physical examinations and laboratory tests failed to detect any abnormalities in humans given various baculoviruses orally [Heimpel, A. M. and Buchanan, L. K., J. Invertebr. Pathol, 9, 55 (1967)].
Baculoviruses are desirable alternatives to conventional chemical insecticides for agricultural pest control because of their selective pathogenicity towards targeted insects, non-pathogenicity towards vertebrates and compatibility with the ecosystem. At least three baculoviruses have been registered by the Environmental Protection Agency for commercial distribution as insecticides: the baculoviruses of Heliothis zea, Orgyia pseudotsugata and Lymantria dispar [Miltenburger, H. G. and. Kreig, A., Advances in Biotechnological Processes, 3, 291 (1984)]. Baculovirus has been successfully tested to control insect populations in Sweden, the Soviet Union, Italy, Canada and the United States [Miltenburger, H. G. and Kreig, A., Advances in Biotechnological Processes, 3, 291 (1984)]. At present, chemical insecticides are the primary means of controlling pest populations. Their use, however, is facing growing opposition-they pollute the environment, and insects may become resistant to their effects. It is estimated that 30% of the agricultural pests in the Western Hemisphere can be controlled by insect viruses [Falcon, L. A., in Viral Pesticides: Present Knowledge and Potential Effects on Public and Environmental Health, Summers, M. D. and Kawanishi, C. Y., eds. (Health Effects Research Laboratory, Office of Health and Ecological Effects, U.S. Environmental Protection Agency, Research Triangle Park, NC, 1978), p.11]. This is a significant figure. Substituting baculovirus for its chemical counterpart in these cases could substantially reduce the total amount of chemical insecticides used in the environment.
Recombinant protein expression in insect cells is achieved byviral infection or stable transformation. For the former, the desired gene is cloned into baculovirus at the site of the wild-type polyhedron gene [Webb, N. R. and Summers, M. D., Technique, 2, 173 (1990); Bishop, D. H. L. and Possee, R. D., Adv. Gene Technol., 1, 55, (1990)]. The polyhedron gene is nonessential for infection or replication of baculovirus. It is the principle component of a protein coat in occlusions which encapsulate virus particles. When a deletion or insertion is made in the polyhedron gene, occlusions fail to form. Occlusion negative viruses produce distinct morphological differences from the wild-type virus. These differences enable a researcher to identify and purify a recombinant virus. In baculovirus, the cloned gene is under the control of the polyhedron promoter, a strong promoter which is responsible for the high expression levels of recombinant protein that characterize this system. Expression of recombinant protein typically begins within 24 hours after viral infection and terminates after 72 hours when the Sf9 culture has lysed.
Stably-transformed insect cells provide an alternate expression system for recombinant protein production [Jarvis, D. L., Fleming, J.-A. G. W., Kovacs, G. R., Summers, M. D., and Guarino, L. A., Biotechnology,, 8, 950 (1990); Cavegn, C., Young, J., Bertrand, M., and Bernard, A. R., in Animal Cell Technology: Products of Today, Prospects for Tomorrow, Spier, R. E., Griffiths, J. B., and Berthold, W., Eds. (Butterworth-Heinemann, Oxford, 1994, pp. 43-49)]. In these cells, the desired gene is expressed continuously in the absence of viral infection. Stable transformation is favored over viral infection when recombinant protein production requires cellular processes that are compromised by the baculovirus. This occurs, for example, in the secretion of recombinant human tissue plasminogen activator from Sf9 cells [Jarvis, D. L., Fleming, J.-A. G. W., Kovacs, G. R., Summers, M. D., and Guarino, L. A., Biotechnology,, 8, 950 (1990)]. Viral infection is favored when the recombinant protein is cytotoxic since protein expression is transient in this system.